Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends

ABSTRACT

The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents in immunotherapy applications. More particularly, the invention provides immunomers for use in methods for generating an immune response or for treating a patient in need of immunostimulation. The immunomers of the invention comprise at least two oligonucleotides linked at their 3′ ends, internucleoside linkages or functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5′ end.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/279,684 filed Oct.24, 2002 which claims the benefit of U.S. Ser. No. 60/344,767 filed Oct.24, 2001, which are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to immunology and immunotherapy applications usingoligonucleotides as immunostimulatory agents.

2. Summary of the Related Art

Oligonucleotides have become indispensable tools in modern molecularbiology, being used in a wide variety of techniques, ranging fromdiagnostic probing methods to PCR to antisense inhibition of geneexpression and immunotherapy applications. This widespread use ofoligonucleotides has led to an increasing demand for rapid, inexpensiveand efficient methods for synthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnosticapplications can now be routinely accomplished. See, e.g., Methods inMolecular Biology , Vol. 20: Protocols for Oligonucleotides and Analogspp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides andAnalogues, A Practical Approach, pp. 87-108 (F. Eckstein, ed., 1991);and Uhlmann and Peyman, supra; Agrawal and Iyer, Curr. Op. in Biotech.6:12 (1995); and Antisense Research and Applications (Crooke and Lebleu,eds., CRC Press, Boca Raton, 1993). Early synthetic approaches includedphosphodiester and phosphotriester chemistries. For example, Khorana etal., J. Molec. Biol. 72:209 (1972) discloses phosphodiester chemistryfor oligonucleotide synthesis. Reese, Tetrahedron Lett. 34:3143-3179(1978), discloses phosphotriester chemistry for synthesis ofoligonucleotides and polynucleotides. These early approaches havelargely given way to the more efficient phosphoramidite andH-phosphonate approaches to synthesis. For example, Beaucage andCaruthers, Tetrahedron Lett. 22:1859-1862 (1981), discloses the use ofdeoxyribonucleoside phosphoramidites in polynucleotide synthesis.Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), disclosesoptimized synthesis of oligonucleotides by the H-phosphonate approach.Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28:3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochem. 23:3443 (1984), discloses synthesisof oligonucleotide phosphorothioates using phosphoramidite chemistry.Jager et al., Biochem. 27:7237 (1988), discloses synthesis ofoligonucleotide phosphoramidates using phosphoramidite chemistry.Agrawal et al., Proc. Natl. Acad. Sci. (USA) 85:7079-7083 (1988),discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

More recently, several researchers have demonstrated the validity of theuse of oligonucleotides as immunostimulatory agents in immunotherapyapplications. The observation that phosphodiester and phosphorothioateoligonucleotides can induce immune stimulation has created interest indeveloping this side effect as a therapeutic tool. These efforts havefocused on phosphorothioate oligonucleotides containing the dinucleotidenatural CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992)teaches that phosphodiester oligonucleotides containing a palindromethat includes a CpG dinucleotide can induce interferon-alpha and gammasynthesis and enhance natural killer activity. Krieg et al., Nature371:546-549 (1995) discloses that phosphorothioate CpG-containingoligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest.98:1119-1129 (1996) discloses that such oligonucleotides activate humanB cells. Moldoveanu et al., Vaccine 16:1216-124 (1998) teaches thatCpG-containing phosphorothioate oligonucleotides enhance immune responseagainst influenza virus. McCluskie and Davis, J. Immunol. 161:4463-4466(1998) teaches that CpG-containing oligonucleotides act as potentadjuvants, enhancing immune response against hepatitis B surfaceantigen.

Other modifications of CpG-containing phosphorothioate oligonucleotidescan also affect their ability to act as modulators of immune response.See, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao etal., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., AntisenseNucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al., Bioorg. Med. Chem.Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000)10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588;Yu et al., Bioorg. Med. Chem. Lett. (2001) 11:2263-2267; and Kandimallaet al., Bioorg. Med. Chem. (2001) 9:807-813.

These reports make clear that there remains a need to be able to enhancethe immune response caused by immunostimulatory oligonucleotides.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for enhancing the immune response causedby oligonucleotide compounds. The methods according to the inventionenable increasing the immunostimulatory effect of immunostimulatoryoligonucleotides for immunotherapy applications. The present inventorshave surprisingly discovered that modification of an immunostimulatoryoligonucleotide to optimally present its 5′ end dramatically enhancesits immunostimulatory capability. Such an oligonucleotide is referred toherein as an “immunomer.”

In a first aspect, therefore, the invention provides immunomerscomprising at least two oligonucleotides linked at their 3′ ends, aninternuceotide linkage, or a functionalized nucleobase or sugar via anon-nucleotidic linker, at least one of the oligonucleotides being animmunostimulatory oligonucleotide and having an accessible 5′ end.

In one embodiment, the immunomer comprises an immunostimulatorydinucleotide of formula 5′-Pyr-Pur-3′, wherein Pyr is a natural ornon-natural pyrimidine nucleoside and Pur is a natural or non-naturalpurine nucleoside.

In another embodiment, the immunomer comprises an immunostimulatorydinucleotide selected from the group consisting of CpG, C*pG, CpG*, andC*pG*, wherein C is cytidine or 2′-deoxycytidine, C* is2′-deoxythymidine, arabinocytidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-O-substitutedarabinocytidine,2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine or other non-natural pyrimidine nucleoside, G isguanosine or 2′-deoxyguanosine, G* is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, 2′-O-substituted-arabinoguanosine, orother non-natural purine nucleoside, and p is an internucleoside linkageselected from the group consisting of phosphodiester, phosphorothioate,and phosphorodithioate. In certain preferred embodiments, theimmunostimulatory dinucleotide is not CpG.

In yet another embodiment, the immunostimulatory oligonucleotidecomprises an immunostimulatory domain of formula (III):5′-Nn-N1-Y-Z-N1-Nn-3′  (III)wherein:

Y is cytidine, 2′-deoxythymidine, 2′ deoxycytidine, arabinocytidine,2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine,2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine,2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine or other non-naturalpyrimidine nucleoside;

Z is guanosine or 2′-deoxyguanosine, G* is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other non-naturalpurine nucleoside

N1, at each occurrence, is preferably a naturally occurring or asynthetic nucleoside or an immunostimulatory moiety selected from thegroup consisting of abasic nucleosides, arabinonucleosides,2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, andnucleosides linked by a phosphodiester or modified internucleosidelinkage to the adjacent nucleoside on the 3′ side, the modifiedinternucleotide linkage being selected from, without limitation, alinker having a length of from about 2 angstroms to about 200 angstroms,C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and phosphorothioate, phosphorodithioate, ormethylphosphonate internucleoside linkage;

Nn, at each occurrence, is a naturally occurring nucleoside or animmunostimulatory moiety, preferably selected from the group consistingof abasic nucleosides, arabinonucleosides, 2′-deoxyuridine,α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, andnucleosides linked by a modified internucleoside linkage to the adjacentnucleoside on the 3′ side, the modified internucleotide linkage beingselected from the group consisting of amino linker, 2′-5′internucleoside linkage, and methylphosphonate internucleoside linkage;

provided that at least one N1 or Nn is an immunostimulatory moiety;

wherein n is a number from 0-30;

wherein the 3′ end, an internucleotide linkage, or a functionalizednucleobase or sugar is linked directly or via a non-nucleotidic linkerto another oligonucleotide, which may or may not be immunostimulatory.

In a second aspect, the invention provides immunomer conjugates,comprising an immunomer, as described above, and an antigen conjugatedto the immunomer at a position other than the accessible 5′ end.

In a third aspect, the invention provides pharmaceutical formulationcomprising an immunomer or an immunomer conjugate according to theinvention and a physiologically acceptable carrier.

In a fourth aspect, the invention provides methods for generating animmune response in a vertebrate, such methods comprising administeringto the vertebrate an immunomer or immunomer conjugate according to theinvention. In some embodiments, the vertebrate is a mammal.

In a fifth aspect, the invention provides methods for therapeuticallytreating a patient having a disease or disorder, such methods comprisingadministering to the patient an immunomer or immunomer conjugateaccording to the invention. In various embodiments, the disease ordisorder to be treated is cancer, an autoimmune disorder, airwayinflammation, asthma, allergy, or a disease caused by a pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NOS 2 and 65) depicts several representative immunomersof the invention.

FIG. 2 depicts several representative immunomers of the invention.

FIG. 3 depicts a group of representative small molecule linkers suitablefor linear synthesis of immumomers of the invention.

FIG. 4 depicts a group of representative small molecule linkers suitablefor parallel synthesis of immunomers of the invention.

FIG. 5 is a synthetic scheme for the linear synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 6 is a synthetic scheme for the parallel synthesis of immunomers ofthe invention. DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 7A is a graphic representation of the induction of IL-12 byimmunomers 1-3 in BALB/c mouse spleen cell cultures. These data suggestthat Immunomer 2, which has accessible 5′-ends, is a stronger inducer ofIL-12 than monomeric Oligo 1, and that Immunomer 3, which does not haveaccessible 5′-ends, has equal or weaker ability to produce immunestimulation compared with oligo 1.

FIG. 7B is a graphic representation of the induction of IL-6 (top tobottom, respectively) by Immunomers 1-3 in BALB/c mouse spleen cellscultures. These data suggest that Immunomer 2, which has accessible5′-ends, is a stronger inducer of IL-6 than monomeric Oligo 1, and thatImmunomer 3, which does not have accessible 5′-ends, has equal or weakerability to induce immune stimulation compared with Oligo 1.

FIG. 7C is a graphic representation of the induction of IL-10 byImmunomers 1-3 (top to bottom, respectively) in BALB/c mouse spleen cellcultures.

FIG. 8A is a graphic representation of the induction of BALB/c mousespleen cell proliferation in cell cultures by different concentrationsof Immunomers 5 and 6, which have inaccessible and accessible 5′-ends,respectively.

FIG. 8B is a graphic representation of BALB/c mouse spleen enlargementby Immunomers 4-6, which have an immunogenic chemical modification inthe 5′-flanking sequence of the CpG motif. Again, the immunomer, whichhas accessible 5′-ends (6), has a greater ability to increase spleenenlargement compared with Immunomer 5, which does not have accessible5′-end and with monomeric Oligo 4.

FIG. 9A is a graphic representation of induction of IL-12 by differentconcentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleencell cultures.

FIG. 9B is a graphic representation of induction of IL-6 by differentconcentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleencell cultures.

FIG. 9C is a graphic representation of induction of IL-10 by differentconcentrations of Oligo 4 and Immunomers 7 and 8 in BALB/c mouse spleencell cultures.

FIG. 10A is a graphic representation of the induction of cellproliferation by Immunomers 14, 15, and 16 in BALB/c mouse spleen cellcultures.

FIG. 10B is a graphic representation of the induction of cellproliferation by IL-12 by different concentrations of Immunomers 14 and16 in BALB/c mouse spleen cell cultures.

FIG. 10C is a graphic representation of the induction of cellproliferation by IL-6 by different concentrations of Immunomers 14 and16 in BALB/c mouse spleen cell cultures.

FIG. 11A is a graphic representation of the induction of cellproliferation by Oligo 4 and 17 and Immunomers 19 and 20 in BALB/c mousespleen cell cultures.

FIG. 11B is a graphic representation of the induction of cellproliferation IL-12 by different concentrations of Oligo 4 and 17 andImmunomers 19 and 20 in BALB/c mouse spleen cell cultures.

FIG. 11C is a graphic representation of the induction of cellproliferation IL-6 by different concentrations of Oligo 4 and 17 andImmunomers 19 and 20 in BALB/c mouse spleen cell cultures.

FIG. 12 is a graphic representation of BALB/c mouse spleen enlargementusing oligonucleotides 4 and immunomers 14, 23, and 24.

FIG. 13 is a schematic representation of the 3′-terminal nucleoside ofan oligonucleotide, showing that a non-nucleotidic linkage can beattached to the nucleoside at the nucleobase, at the 3′ position, or atthe 2′ position.

FIG. 14 shows the chemical substitutions used in Example 13.

FIG. 15 shows cytokine profiles obtained using the modifiedoligonucleotides of Example 13.

FIG. 16 shows relative cytokine induction for glycerol linkers comparedwith amino linkers.

FIG. 17 shows relative cytokine induction for various linkers and linkercombinations.

FIGS. 18 A-E shows relative nuclease resistance for various PS and POimmunomers and oligonucleotides.

FIG. 19 shows relative cytokine induction for PO immunomers comparedwith PS immunomers in BALB/c mouse spleen cell cultures.

FIG. 20 shows relative cytokine induction for PO immunomers comparedwith PS immunomers in C3H/Hej mouse spleen cell cultures.

FIG. 21 shows relative cytokine induction for PO immunomers comparedwith PS immunomers in C3H/Hej mouse spleen cell cultures at highconcentrations of immunomers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of oligonucleotides asimmunostimulatory agents for immunotherapy applications. The issuedpatents, patent applications, and references that are cited herein arehereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.In the event of inconsistencies between any teaching of any referencecited herein and the present specification, the latter shall prevail forpurposes of the invention.

The invention provides methods for enhancing the immune response causedby immunostimulatory compounds used for immunotherapy applications suchas, but not limited to, treatment of cancer, autoimmune disorders,asthma, respiratory allergies, food allergies, and bacteria, parasitic,and viral infections in adult and pediatric human and veterinaryapplications. Thus, the invention further provides compounds havingoptimal levels of immunostimulatory effect for immunotherapy and methodsfor making and using such compounds. In addition, immunomers of theinvention are useful as adjuvants in combination with DNA vaccines,antibodies, allergens, chemotherapeutic agents, and antisenseoligonucleotides.

The present inventors have surprisingly discovered that modification ofan immunostimulatory oligonucleotide to optimally present its 5′ endsdramatically affects its immunostimulatory capabilities. Such anoligonucleotide is referred to herein as an “immunomer.”

In a first aspect, the invention provides immunomers comprising at leasttwo oligonucleotides linked at their 3′ ends, or an internucleosidelinkage or a functionalized nucleobase or sugar to a non-nucleotidiclinker, at least one of the oligonucleotides being an immunostimulatoryoligonucleotide and having an accessible 5′ end. As used herein, theterm “accessible 5′ end” means that the 5′ end of the oligonucleotide issufficiently available such that the factors that recognize and bind toimmunomers and stimulate the immune system have access to it. Inoligonucleotides having an accessible 5′ end, the 5′ OH position of theterminal sugar is not covalently linked to more than two nucleosideresidues. Optionally, the 5′ OH can be linked to a phosphate,phosphorothioate, or phosphorodithioate moiety, an aromatic or aliphaticlinker, cholesterol, or another entity which does not interfere withaccessibility.

For purposes of the invention, the term “immunomer” refers to anycompound comprising at least two oligonucleotides linked at their 3′ends or internucleoside linkages, or functionalized nucleobase or sugardirectly or via a non-nucleotidic linker, at least one of theoligonucleotides (in the context of the immunomer) being animmunostimulatory oligonucleotide and having an accessible 5′ end,wherein the compound induces an immune response when administered to avertebrate. In some embodiments, the vertebrate is a mammal, including ahuman.

In some embodiments, the immunomer comprises two or moreimmunostimulatory oligonucleotides, (in the context of the immunomer)which may be the same or different. Preferably, each suchimmunostimulatory oligonucleotide has at least one accessible 5′ end.

In certain embodiments, in addition to the immunostimulatoryoligonucleotide(s), the immunomer also comprises at least oneoligonucleotide that is complementary to a gene. As used herein, theterm “complementary to” means that the oligonucleotide hybridizes underphysiological conditions to a region of the gene. In some embodiments,the oligonucleotide downregulates expression of a gene. Suchdownregulatory oligonucleotides preferably are selected from the groupconsisting of antisense oligonucleotides, ribozyme oligonucleotides,small inhibitory RNAs and decoy oligonucleotides. As used herein, theterm “downregulate a gene” means to inhibit the transcription of a geneor translation of a gene product. Thus, the immunomers according tothese embodiments of the invention can be used to target one or morespecific disease targets, while also stimulating the immune system.

In certain embodiments, the immunomer includes a ribozyme or a decoyoligonucleotide. As used herein, the term “ribozyme” refers to anoligonucleotide that possesses catalytic activity. Preferably, theribozyme binds to a specific nucleic acid target and cleaves the target.As used herein, the term “decoy oligonucleotide” refers to anoligonucleotide that binds to a transcription factor in asequence-specific manner and arrests transcription activity. Preferably,the ribozyme or decoy oligonucleotide exhibits secondary structure,including, without limitation, stem-loop or hairpin structures. Incertain embodiments, at least one oligonucleotide comprisingpoly(I)-poly(dC). In certain embodiments, at least one set of Nnincludes a string of 3 to 10 dGs and/or Gs or 2′-substituted ribo orarabino Gs.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked nucleoside units. Sucholigonucleotides can be obtained from existing nucleic acid sources,including genomic or cDNA, but are preferably produced by syntheticmethods. In preferred embodiments each nucleoside unit includes aheterocyclic base and a pentofuranosyl, trehalose, arabinose,2′-deoxy-2′-substitutedarabinose, 2′-O-substitutedarabinose or hexosesugar group. The nucleoside residues can be coupled to each other by anyof the numerous known internucleoside linkages. Such internucleosidelinkages include, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleoside linkages. The term“oligonucleotide” also encompasses polynucleosides having one or morestereospecific internucleoside linkage (e.g., (R_(p))- or(S_(p))-phosphorothioate, alkylphosphonate, or phosphotriesterlinkages). As used herein, the terms “oligonucleotide” and“dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphorothioate, or phosphorodithioate linkages, or combinationsthereof.

In some embodiments, the oligonucleotides each have from about 3 toabout 35 nucleoside residues, preferably from about 4 to about 30nucleoside residues, more preferably from about 4 to about 20 nucleosideresidues. In some embodiments, the oligonucleotides have from about 5 toabout 18, or from about 5 to about 14, nucleoside residues. As usedherein, the term “about” implies that the exact number is not critical.Thus, the number of nucleoside residues in the oligonucleotides is notcritical, and oligonucleotides having one or two fewer nucleosideresidues, or from one to several additional nucleoside residues arecontemplated as equivalents of each of the embodiments described above.In some embodiments, one or more of the oligonucleotides have 11nucleotides.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The oligonucleotides of the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-fluoroarabinoside.

For purposes of the invention, the term “2′-substituted ribonucleoside”includes ribonucleosides in which the hydroxyl group at the 2′ positionof the pentose moiety is substituted to produce a 2′-O-substitutedribonucleoside. Preferably, such substitution is with a lower alkylgroup containing 1-6 saturated or unsaturated carbon atoms, or with anaryl group having 6-10 carbon atoms, wherein such alkyl, or aryl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carboalkoxy, or amino groups. Examples of such 2′-O-substitutedribonucleosides include, without limitation 2′-O-methylribonucleosidesand 2′-O-methoxyethylribonucleosides.

The term “2′-substituted ribonucleoside” also includes ribonucleosidesin which the 2′-hydroxyl group is replaced with a lower alkyl groupcontaining 1-6 saturated or unsaturated carbon atoms, or with an aminoor halo group. Examples of such 2′-substituted ribonucleosides include,without limitation, 2′-amino, 2′-fluoro, 2′-allyl, and 2′-propargylribonucleosides.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage. One preferredexample of such a chimeric oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and non-ionic linkages such as alkylphosphonate oralkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos.5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One preferred example of such a hybridoligonucleotide comprises a ribonucleotide or 2′-substitutedribonucleotide region, and a deoxyribonucleotide region (see, e.g.,Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

For purposes of the invention, the term “immunostimulatoryoligonucleotide” refers to an oligonucleotide as described above thatinduces an immune response when administered to a vertebrate, such as afish, fowl, or mammal. As used herein, the term “mammal” includes,without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs,rabbits, non-human primates, and humans. Useful immunostimulatoryoligonucleotides can be found described in Agrawal et al., WO 98/49288,published Nov. 5, 1998; WO 01/12804, published Feb. 22, 2001; WO01/55370, published Aug. 2, 2001; PCT/US01/13682, filed Apr. 30, 2001;and PCT/US01/30137, filed Sep. 26, 2001. Preferably, theimmunostimulatory oligonucleotide comprises at least one phosphodiester,phosphorothioate, or phosphordithioate internucleoside linkage.

In some embodiments, the immunostimulatory oligonucleotide comprises animmunostimulatory dinucleotide of formula 5′-Pyr-Pur-3′, wherein Pyr isa natural or synthetic pyrimidine nucleoside and Pur is a natural orsynthetic purine nucleoside. As used herein, the term “pyrimidinenucleoside” refers to a nucleoside wherein the base component of thenucleoside is a pyrimidine base. Similarly, the term “purine nucleoside”refers to a nucleoside wherein the base component of the nucleoside is apurine base. For purposes of the invention, a “synthetic” pyrimidine orpurine nucleoside includes a non-naturally occurring pyrimidine orpurine base, a non-naturally occurring sugar moiety, or a combinationthereof.

Preferred pyrimidine nucleosides according to the invention have thestructure (I):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, hydrogen bond acceptor, hydrophilic group, hydrophobic group,electron withdrawing group and electron donating group;

A is a hydrogen bond acceptor or a hydrophilic group;

A′ is selected from the group consisting of hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group;

X is carbon or nitrogen; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, and the ring nitrogen atoms of an aromaticheterocycle, e.g., N3 of cytosine.

In some embodiments, the base moiety in (I) is a non-naturally occurringpyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,and 4-thiouracil. However, in some embodiments 5-bromocytosine isspecifically excluded.

In some embodiments, the sugar moiety S′ in (I) is a non-naturallyoccurring sugar moiety. For purposes of the present invention, a“naturally occurring sugar moiety” is a sugar moiety that occursnaturally as part of nucleic acid, e.g., ribose and 2′-deoxyribose, anda “non-naturally occurring sugar moiety” is any sugar that does notoccur naturally as part of a nucleic acid, but which can be used in thebackbone for an oligonucleotide, e.g, hexose. Arabinose and arabinosederivatives are examples of a preferred sugar moieties.

Preferred purine nucleoside analogs according to the invention have thestructure (II):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, and hydrophilic group;

A is a hydrogen bond acceptor or a hydrophilic group;

X is carbon or nitrogen;

each L is independently selected from the group consisting of C, O, Nand S; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, —NO₂ and the ring nitrogen atoms of an aromaticheterocycle, e.g., N1 of guanine.

In some embodiments, the base moiety in (II) is a non-naturallyoccurring purine base. Examples of preferred non-naturally occurringpurine bases include, without limitation, 6-thioguanine and7-deazaguanine. In some embodiments, the sugar moiety S′ in (II) is anaturally occurring sugar moiety, as described above for structure (I).

In preferred embodiments, the immunostimulatory dinucleotide is selectedfrom the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C iscytidine or 2′-deoxycytidine, C* is 2′-deoxythymidine, arabinocytidine,2′-deoxythymidine, 2′-deoxy-2′-substitutedarabinocytidine,2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine,2′-deoxy-N4-alkyl-cytidine, 2′-deoxy4-thiouridine or other non-naturalpyrimidine nucleoside, G is guanosine or 2′-deoxyguanosine, G* is 2′deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other non-naturalpurine nucleoside, and p is an internucleoside linkage selected from thegroup consisting of phosphodiester, phosphorothioate, andphosphorodithioate. In certain preferred embodiments, theimmunostimulatory dinucleotide is not CpG.

The immunostimulatory oligonucleotides may include immunostimulatorymoieties on one or both sides of the immunostimulatory dinucleotide.Thus, in some embodiments, the immunostimulatory oligonucleotidecomprises in immunostimulatory domain of structure (III):5′-Nn-N1-Y-Z-N1-Nn-3′  (III)

wherein:

Y is cytidine, 2′deoxythymidine, 2′ deoxycytidine arabinocytidine,2′-deoxy-2′-substitutedarabinocytidine, 2′-deoxythymidine,2′-O-substitutedarabinocytidine, 2′-eoxy-5-hydroxycytidine,2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine or other non-naturalpyrimidine nucleoside;

Z is guanosine or 2′-deoxyguanosine, G* is 2′ deoxy-7-deazaguanosine,2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′deoxyinosine, or other non-naturalpurine nucleoside;

N1, at each occurrence, is preferably a naturally occurring or asynthetic nucleoside or an immunostimulatory moiety selected from thegroup consisting of abasic nucleosides, arabinonucleosides,2′-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, andnucleosides linked by a phosphodiester or modified internucleosidelinkage to the adjacent nucleoside on the 3′ side, the modifiedinternucleotide linkage being selected from, without limitation, alinker having a length of from about 2 angstroms to about 200 angstroms,C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and phosphorothioate, phosphorodithioate, ormethylphosphonate internucleoside linkage;

Nn, at each occurrence, is preferably a naturally occurring nucleosideor an immunostimulatory moiety selected from the group consisting ofabasic nucleosides, arabinonucleosides, 2′-deoxyuridine,α-deoxyribonucleosides, 2′-O-substituted ribonucleosides, andnucleosides linked by a modified internucleoside linkage to the adjacentnucleoside on the 3′ side, the modified internucleotide linkagepreferably being selected from the group consisting of amino linker,2′-5′ internucleoside linkage, and methylphosphonate internucleosidelinkage;

provided that at least one N1 or Nn is an immunostimulatory moiety;

wherein n is a number from 0 to 30; and

wherein the 3′end, an internucleoside linker, or a derivatizednucleobase or sugar is linked directly or via a non-nucleotidic linkerto another oligonucleotide, which may or may not be immunostimulatory.

In some preferred embodiments, YZ is arabinocytidine or2′-deoxy-2′-substituted arabinocytidine and arabinoguanosine or2′deoxy-2′-substituted arabinoguanosine. Preferred immunostimulatorymoieties include modifications in the phosphate backbones, including,without limitation, methylphosphonates, methylphosphonothioates,phosphotriesters, phosphothiotriesters, phosphorothioates,phosphorodithioates, triester prodrugs, sulfones, sulfonamides,sulfamates, formacetal, N-methylhydroxylamine, carbonate, carbamate,morpholino, boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (R_(p))- or (S_(p))-phosphorothioate,alkylphosphonate, or phosphotriester linkages).

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having sugar modifications, including, withoutlimitation, 2′-substituted pentose sugars including, without limitation,2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, including,without limitation, 3′-O-methylribose; 1′,2′-dideoxyribose; arabinose;substituted arabinose sugars, including, without limitation,1′-methylarabinose, 3′-hydroxymethylarabinose,4′-hydroxymethyl-arabinose, and 2′-substituted arabinose sugars; hexosesugars, including, without limitation, 1,5-anhydrohexitol; andalpha-anomers. In embodiments in which the modified sugar is a3′-deoxyribonucleoside or a 3′-O-substituted ribonucleoside, theimmunostimulatory moiety is attached to the adjacent nucleoside by wayof a 2′-5′ internucleoside linkage.

Preferred immunostimulatory moieties according to the invention furtherinclude oligonucleotides having other carbohydrate backbonemodifications and replacements, including peptide nucleic acids (PNA),peptide nucleic acids with phosphate groups (PHONA), locked nucleicacids (LNA), morpholino backbone oligonucleotides, and oligonucleotideshaving backbone linker sections having a length of from about 2angstroms to about 200 angstroms, including without limitation, alkyllinkers or amino linkers. The alkyl linker may be branched orunbranched, substituted or unsubstituted, and chirally pure or a racemicmixture. Most preferably, such alkyl linkers have from about 2 to about18 carbon atoms. In some preferred embodiments such alkyl linkers havefrom about 3 to about 9 carbon atoms. Some alkyl linkers include one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthioether. Some such functionalized alkyl linkers are poly(ethyleneglycol) linkers of formula —O—(CH₂—CH₂—O—)_(n) (n=1-9). Some otherfunctionalized alkyl linkers are peptides or amino acids.

Preferred immunostimulatory moieties according to the invention furtherinclude DNA isoforms, including, without limitation,β-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferredimmunostimulatory moieties according to the invention incorporate 3′modifications, and further include nucleosides having unnaturalinternucleoside linkage positions, including, without limitation, 2′-5′,2′-2′, 3′-3′ and 5′-5′ linkages.

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having modified heterocyclic bases, including,without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine,N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil,6-thioguanine, 7-deazaguanine, inosine, nitropyrrole,C5-propynylpyrimidine, and diamninopurines, including, withoutlimitation, 2,6-diaminopurine.

By way of specific illustration and not by way of limitation, forexample, in the immunostimulatory domain of structure (III), amethylphosphonate internucleoside linkage at position N1 or Nn is animmunostimulatory moiety, a linker having a length of from about 2angstroms to about 200 angstroms, C2-C18 alkyl linker at position X1 isan immunostimulatory moiety, and a β-L-deoxyriboiucleoside at positionX1 is an immunostimulatory moiety. See Table 1 below for representativepositions and structures of immunostimulatory moieties. It is to beunderstood that reference to a linker as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis substituted at its 3′-hydroxyl with the indicated linker, therebycreating a modified internucleoside linkage between that nucleosideresidue and the adjacent nucleoside on the 3′ side. Similarly, referenceto a modified internucleoside linkage as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis linked to the adjacent nucleoside on the 3′ side by way of therecited linkage.

TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES N1Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside,2′-deoxyuridine, β-L- deoxyribonucleoside C2-C18 alkyl linker,poly(ethylene glycol) linkage, 2-aminobutyl-1,3-propanediol linker(amino linker), 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage Nn Naturally-occurring nucleosides, abasicnucleoside, arabinonucleosides, 2′-deoxyuridine, 2′-O-substitutedribonucleoside, 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage, provided that N1 and N2 cannot both be abasiclinkages

Table 2 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving an upstream potentiation domain. As used herein, the term “Spacer9” refers to a poly(ethylene glycol) linker of formula—O—(CH₂CH₂—O)_(n)—, wherein n is 3. The term “Spacer 18” refers to apoly(ethylene glycol) linker of formula —O—(CH₂CH₂—O)_(n)—, wherein n is6. As used herein, the term “C2-C18 alkyl linker refers to a linker offormula —O—(CH₂)_(q)—O—, where q is an integer from 2 to 18.Accordingly, the terms “C3-linker” and “C3-alkyl linker” refer to alinker of formula —O—(CH₂)₃—O—. For each of Spacer 9, Spacer 18, andC2-C18 alkyl linker, the linker is connected to the adjacent nucleosidesby way of phosphodiester, phosphorothioate, or phosphorodithioatelinkages.

TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol linker 5′N1 Naturally-occurring nucleosides, β-L-deoxyribonucleoside, C2-C18alkyl linker, poly(ethylene glycol), abasic linker,2-aminobutyl-1,3-propanediol linker 3′ N1 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, 2′-O-methyl- ribonucleoside, C2-C18alkyl linker, Spacer 9, Spacer 18 3′ N2 Naturally-occurring nucleosides,1′,2′-dideoxyribose, 3′- deoxyribonucleoside, β-L-deoxyribonucleoside,2′-O-propargyl- ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage 3′ N 3 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage, 2′-5′ internucleosidelinkage, d(G)n, polyI-polydC 3′N2 + 3′N 3 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, d(G)n, polyI-polydC 3′N3 +3′ N 4 2′-O-methoxyethyl-ribonucleoside, methylphosphonateinternucleoside linkage, d(G)n, polyI-polydC 3′N5 + 3′ N 61′,2′-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyI-polydC 5′N1 + 3′N 3 1′,2′-dideoxyribose, d(G)n, polyI-polydC

Table 3 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving a downstream potentiation domain.

TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2methylphosphonate internucleoside linkage 5′ N1 methylphosphonateinternucleoside linkage 3′ N1 1′,2′-dideoxyribose, methylphosphonateinternucleoside linkage, 2′-O-methyl 3′ N2 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18,2-aminobutyl-1,3- propanediol linker, methylphosphonate internucleosidelinkage, 2′-O-methyl 3′ N3 3′-deoxyribonucleoside, 3′-O-substitutedribonucleoside, 2′-O-propargyl-ribonucleoside 3′N2 + 3′ N31′,2′-dideoxyribose, β-L-deoxyribonucleoside

The immunomers according to the invention comprise at least twooligonucleotides linked at their 3′ ends or internucleoside linkage or afunctionalized nucleobase or sugar via a non-nucleotidic linker. Forpurposes of the invention, a “non-nucleotidic linker” is any moiety thatcan be linked to the oligonucleotides by way of covalent or non-covalentlinkages. Preferably such linker is from about 2 angstroms to about 200angstroms in length. Several examples of preferred linkers are set forthbelow. Non-covalent linkages include, but are not limited to,electrostatic interaction, hydrophobic interactions, π-stackinginteractions, and hydrogen bonding. The term “non-nucleotidic linker” isnot meant to refer to an internucleoside linkage, as described above,e.g., a phosphodiester, phosphorothioate, or phosphorodithioatefunctional group, that directly connects the 3′-hydroxyl groups of twonucleosides. For purposes of this invention, such a direct 3′-3′ linkageis considered to be a “nucleotidic linkage.”

In some embodiments, the non-nucleotidic linker is a metal, including,without limitation, gold particles. In some other embodiments, thenon-nucleotidic linker is a soluble or insoluble biodegradable polymerbead.

In yet other embodiments, the non-nucleotidic linker is an organicmoiety having functional groups that permit attachment to theoligonucleotide. Such attachment preferably is by any stable covalentlinkage. As a non-limiting example, the linker may be attached to anysuitable position on the nucleoside, as illustrated in FIG. 13. In somepreferred embodiments, the linker is attached to the 3′-hydroxyl. Insuch embodiments, the linker preferably comprises a hydroxyl functionalgroup, which preferably is attached to the 3′-hydroxyl by means of aphosphodiester, phosphorothioate, phosphorodithioate ornon-phosphate-based linkages.

In some embodiments, the non-nucleotidic linker is a biomolecule,including, without limitation, polypeptides, antibodies, lipids,antigens, allergens, and oligosaccharides. In some other embodiments,the non-nucleotidic linker is a small molecule. For purposes of theinvention, a small molecule is an organic moiety having a molecularweight of less than 1,000 Da. In some embodiments, the small moleculehas a molecular weight of less than 750 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligonucleotides or appended to it, one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthiourea. The small molecule can be cyclic or acyclic. Examples of smallmolecule linkers include, but are not limited to, amino acids,carbohydrates, cyclodextrins, adamantane, cholesterol, haptens andantibiotics. However, for purposes of describing the non-nucleotidiclinker, the term “small molecule” is not intended to include anucleoside.

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6, or from 2 to about 4.

Some non-nucleotidic linkers according to the invention permitattachment of more than two oligonucleotides, as schematically depictedin FIG. 1. For example, the small molecule linker glycerol has threehydroxyl groups to which oligonucleotides may be covalently attached.Some immunomers according to the invention, therefore, comprise morethan two oligonucleotides linked at their 3′ ends to a non-nucleotidiclinker. Some such immunomers comprise at least two immunostimulatoryoligonucleotides, each having an accessible 5′ end.

The immunomers of the invention may conveniently be synthesized using anautomated synthesizer and phosphoramidite approach as schematicallydepicted in FIGS. 5 and 6, and further described in the Examples. Insome embodiments, the immunomers are synthesized by a linear synthesisapproach (see FIG. 5). As used herein, the term “linear synthesis”refers to a synthesis that starts at one end of the immunomer andprogresses linearly to the other end. Linear synthesis permitsincorporation of either identical or un-identical (in terms of length,base composition and/or chemical modifications incorporated) monomericunits into the immunomers.

An alternative mode of synthesis is “parallel synthesis”, in whichsynthesis proceeds outward from a central linker moiety (see FIG. 6). Asolid support attached linker can be used for parallel synthesis, as isdescribed in U.S. Pat. No. 5,912,332. Alternatively, a universal solidsupport (such as phosphate attached controlled pore glass support can beused.

Parallel synthesis of immunomers has several advantages over linearsynthesis: (1) parallel synthesis permits the incorporation of identicalmonomeric units; (2) unlike in linear synthesis, both (or all) themonomeric units are synthesized at the same time, thereby the number ofsynthetic steps and the time required for the synthesis is the same asthat of a monomeric unit; and (3) the reduction in synthetic stepsimproves purity and yield of the final immunomer product.

At the end of the synthesis by either linear synthesis or parallelsynthesis protocols, the immunomers may conveniently be deprotected withconcentrated ammonia solution or as recommended by the phosphoramiditesupplier, if a modified nucleoside is incorporated. The productimmunomer is preferably purified by reversed phase HPLC, detritylated,desalted and dialyzed.

Table 4 shows representative immunomers according to the invention.Additional immunomers are found described in the Examples.

TABLE 4 Examples of Immunomer Sequences Oligo or Immunomer No. Sequencesand Modification (5′-3′)  1 (SEQ ID NO: 1) 5′-GAGAACGCTCGACCTT-3′  2(SEQ ID NO: 1) 5′-GAGAACGCTCGACCTT-3′-3′-TTCCAGCTCGCAAGAG-5′  3 (SEQ IDNO: 1) 3′-TTCCAGCTCGCAAGAG-5′-5′-GAGAACGCTCGACCTT-3′  4 (SEQ ID NO: 2)5′-CTATCTGACGTTCTCTGT-3′  5 (SEQ ID NO: 3)

 6 (SEQ ID NO: 3)

 7 (SEQ ID NO: 2)

 8 (SEQ ID NO: 2)

 9 (SEQ ID NO: 4)

10 (SEQ ID NO: 5)

11 (SEQ ID NO: 6)

12 (SEQ ID NO: 7)

13 (SEQ ID NO: 8) 5′-CTGACGTTCTCTGT-3′ 14 (SEQ ID NO: 8)

15 (SEQ ID NO: 6)

16 (SEQ ID NO: 7)

17 (SEQ ID NO: 9) 5′-XXTGACGTTCTCTGT-3′ 18 (SEQ ID NO: 10)

19 (SEQ ID NO: 11)

20 (SEQ ID NO: 12)

21 (SEQ ID NO: 13) 5′-TCTGACGTTCT-3′ 22 (SEQ ID NO: 14)

23 (SEQ ID NO: 15)

24 (SEQ ID NO: 16)

L = C3-alkyl linker; X = 1′,2′-dideoxyriboside; Y = ^(50H)dC; R =7-deaza-dG

In a second aspect, the invention provides immunomer conjugates,comprising an immunomer, as described above, and an antigen conjugatedto the immunomer at a position other than the accessible 5′ end. In someembodiments, the non-nucleotidic linker comprises an antigen, which isconjugated to the oligonucleotide. In some other embodiments, theantigen is conjugated to the oligonucleotide at a position other thanits 3′ end. In some embodiments, the antigen produces a vaccine effect.

The antigen is preferably selected from the group consisting of antigensassociated with a pathogen, antigens associated with a cancer, antigensassociated with an auto-immune disorder, and antigens associated withother diseases such as, but not limited to, veterinary or pediatricdiseases. For purposes of the invention, the term “associated with”means that the antigen is present when the pathogen, cancer, auto-immunedisorder, food allergy, respiratory allergy, asthma or other disease ispresent, but either is not present, or is present in reduced amounts,when the pathogen, cancer, auto-immune disorder, food allergy,respiratory allergy, or disease is absent.

The immunomer is covalently linked to the antigen, or it is otherwiseoperatively associated with the antigen. As used herein, the term“operatively associated with” refers to any association that maintainsthe activity of both immunomer and antigen. Nonlimiting examples of suchoperative associations include being part of the same liposome or othersuch delivery vehicle or reagent. In embodiments wherein the immunomeris covalently linked to the antigen, such covalent linkage preferably isat any position on the immunomer other than an accessible 5′ end of animmunostimulatory oligonucleotide. For example, the antigen may beattached at an internucleoside linkage or may be attached to thenon-nucleotidic linker. Alternatively, the antigen may itself be thenon-nucleotidic linker.

In a third aspect, the invention provides pharmaceutical formulationscomprising an immunomer or immunomer conjugate according to theinvention and a physiologically acceptable carrier. As used herein, theterm “physiologically acceptable” refers to a material that does notinterfere with the effectiveness of the immunomer and is compatible witha biological system such as a cell, cell culture, tissue, or organism.Preferably, the biological system is a living organism, such as avertebrate.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990.

In a fourth aspect, the invention provides methods for generating animmune response in a vertebrate, such methods comprising administeringto the vertebrate an immunomer or immunomer conjugate according to theinvention. In some embodiments, the vertebrate is a mammal. For purposesof this invention, the term “mammal” is expressly intended to includehumans. In preferred embodiments, the immunomer or immunomer conjugateis administered to a vertebrate in need of immunostimulation.

In the methods according to this aspect of the invention, administrationof immunomers can be by any suitable route, including, withoutlimitation, parenteral, oral, sublingual, transdermal, topical,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,by gene gun, dermal patch or in eye drop or mouthwash form.Administration of the therapeutic compositions of immunomers can becarried out using known procedures at dosages and for periods of timeeffective to reduce symptoms or surrogate markers of the disease. Whenadministered systemically, the therapeutic composition is preferablyadministered at a sufficient dosage to attain a blood level of immunomerfrom about 0.0001 micromolar to about 10 micromolar. For localizedadministration, much lower concentrations than this may be effective,and much higher concentrations may be tolerated. Preferably, a totaldosage of immunomer ranges from about 0.001 mg per patient per day toabout 200 mg per kg body weight per day. It may be desirable toadminister simultaneously, or sequentially a therapeutically effectiveamount of one or more of the therapeutic compositions of the inventionto an individual as a single treatment episode.

In certain preferred embodiments, immunomers according to the inventionare administered in combination with vaccines, antibodies, cytotoxicagents, allergens, antibiotics, antisense oligonucleotides, peptides,proteins, gene therapy vectors, DNA vaccines and/or adjuvants to enhancethe specificity or magnitude of the immune response. In theseembodiments, the immunomers of the invention can variously act asadjuvants and/or produce direct immunostimulatory effects.

Either the immunomer or the vaccine, or both, may optionally be linkedto an immunogenic protein, such as keyhole limpet hemocyanin (KLH),cholera toxin B subunit, or any other immunogenic carrier protein. Anyof the plethora of adjuvants may be used including, without limitation,Freund's complete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, andsaponins, including QS-21, imiquimod, R848, or combinations thereof.

For purposes of this aspect of the invention, the term “in combinationwith” means in the course of treating the same disease in the samepatient, and includes administering the immunomer and/or the vaccineand/or the adjuvant in any order, including simultaneous administration,as well as temporally spaced order of up to several days apart. Suchcombination treatment may also include more than a single administrationof the immunomer, and/or independently the vaccine, and/or independentlythe adjuvant. The administration of the immunomer and/or vaccine and/oradjuvant may be by the same or different routes.

The methods according to this aspect of the invention are useful formodel studies of the immune system. The methods are also useful for theprophylactic or therapeutic treatment of human or animal disease. Forexample, the methods are useful for pediatric and veterinary vaccineapplications.

In a fifth aspect, the invention provides methods for therapeuticallytreating a patient having a disease or disorder, such methods comprisingadministering to the patient an immunomer or immunomer conjugateaccording to the invention. In various embodiments, the disease ordisorder to be treated is cancer, an autoimmune disorder, airwayinflammation, inflammatory disorders, allergy, asthma or a diseasecaused by a pathogen. Pathogens include bacteria, parasites, fungi,viruses, viroids and prions. Administration is carried out as describedfor the fourth aspect of the invention.

For purposes of the invention, the term “allergy” includes, withoutlimitation, food allergies and respiratory allergies. The term “airwayinflammation” includes, without limitation, asthma. As used herein, theterm “autoimmune disorder” refers to disorders in which “self” proteinsundergo attack by the immune system. Such term includes autoimmuneasthma.

In any of the methods according to this aspect of the invention, theimmunomer or immunomer conjugate can be administered in combination withany other agent useful for treating the disease or condition that doesnot diminish the immunostimulatory effect of the immunomer. For example,in the treatment of cancer, it is contemplated that the immunomer orimmunomer conjugate may be administered in combination with achemotherapeutic compound.

The examples below are intended to further illustrate certain preferredembodiments of the invention, and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides ContainingImmunomodulatory Moieties

Oligonucleotides were synthesized on a 1 μmol scale using an automatedDNA synthesizer (Expedite 8909; PerSeptive Biosystems, Framingham,Mass.), following the linear synthesis or parallel synthesis proceduresoutlined in FIGS. 5 and 6.

Deoxyribonucleoside phosphoramidites were obtained from AppliedBiosystems (Foster City, Calif.). 1′,2′-dideoxyribose phosphoramidite,propyl-1-phosphoramidite, 2-deoxyuridine phosphoramidite,1,3-bis-[5-(4,4′-dimethoxytrityl)pentylamidyl]-2-propanolphosphoramidite and methyl phosponamidite were obtained from GlenResearch (Sterling, Va.). β-L-2′-deoxyribonucleoside phosphoramidite,α-2′-deoxyribonucleoside phosphoramidite, mono-DMT-glycerolphosphoramidite and di-DMT-glycerol phosphoramidite were obtained fromChemGenes (Ashland, Mass.). (4-Aminobutyl)-1,3-propanediolphosphoramidite was obtained from Clontech (Palo Alto, Calif.).Arabinocytidine phosphoramidite, arabinoguanosine, arabinothymidine andarabinouridine were obtained from Reliable Pharmaceutical (St. Louis,Mo.). Arabinoguanosine phosphoramidite, arabinothymidine phosphoramiditeand arabinouridine phosphoramidite were synthesized at Hybridon, Inc.(Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062).

All nucleoside phosphoramidites were characterized by ³¹P and ¹H NMRspectra. Modified nucleosides were incorporated at specific sites usingnormal coupling cycles. After synthesis, oligonucleotides weredeprotected using concentrated ammonium hydroxide and purified byreverse phase HPLC, followed by dialysis. Purified oligonucleotides assodium salt form were lyophilized prior to use. Purity was tested by CGEand MALDI-TOF MS.

Example 2 Analysis of Spleen Cell Proliferation

In vitro analysis of splenocyte proliferation was carried out usingstandard procedures as described previously (see, e.g., Zhao et al.,Biochem Pharma 51:173-182 (1996)). The results are shown in FIG. 8A.These results demonstrate that at the higher concentrations, Immunomer6, having two accessible 5′ ends results in greater splenocyteproliferation than does Immunomer 5, having no accessible 5′ end orOligonucleotide 4, with a single accessible 5′ end. Immunomer 6 alsocauses greater splenocyte proliferation than the LPS positive control.

Example 3 In vivo Splenomegaly Assays

To test the applicability of the in vitro results to an in vivo model,selected oligonucleotides were administered to mice and the degree ofsplenomegaly was measured as an indicator of the level ofimmunostimulatory activity. A single dose of 5 mg/kg was administered toBALB/c mice (female, 4-6 weeks old, Harlan Sprague Dawley Inc, Baltic,Conn.) intraperitoneally. The mice were sacrificed 72 hours afteroligonucleotide administration, and spleens were harvested and weighed.The results are shown in FIG. 8B. These results demonstrate thatImmunomer 6, having two accessible 5′ ends, has a far greaterimmunostimulatory effect than do Oligonucleotide 4 or Immunomer 5.

Example 4 Cytokine Analysis

The secretion of IL-12 and IL-6 in vertebrate cells, preferably BALB/cmouse spleen cells or human PBMC, was measured by sandwich ELISA. Therequired reagents including cytokine antibodies and cytokine standardswere purchased form PharMingen, San Diego, Calif. ELISA plates (Costar)were incubated with appropriate antibodies at 5 μg/mL in PBSN buffer(PBS/0.05% sodium azide, pH 9.6) overnight at 4° C. and then blockedwith PBS/1% BSA at 37 ° C. for 30 minutes. Cell culture supernatants andcytokine standards were appropriately diluted with PBS/10% FBS, added tothe plates in triplicate, and incubated at 25° C. for 2 hours. Plateswere overlaid with 1 μg/mL appropriate biotinylated antibody andincubated at 25° C. for 1.5 hours. The plates were then washedextensively with PBS-T Buffer (PBS/0.05% Tween 20) and further incubatedat 25° C. for 1.5 hours after adding streptavidin conjugated peroxidase(Sigma, St. Louis, Mo.). The plates were developed with Sure Blue™(Kirkegaard and Perry) chromogenic reagent and the reaction wasterminated by adding Stop Solution (Kirkegaard and Perry). The colorchange was measured on a Ceres 900 HDI Spectrophotometer (Bio-TekInstruments). The results are shown in Table 5A below.

Human peripheral blood mononuclear cells (PBMCs) were isolated fromperipheral blood of healthy volunteers by Ficoll-Paque density gradientcentrifugation (Histopaque-1077, Sigma, St. Louis, Mo.). Briefly,heparinized blood was layered onto the Histopaque-1077 (equal volume) ina conical centrifuge and centrifuged at 400×g for 30 minutes at roomtemperature. The buffy coat, containing the mononuclear cells, wasremoved carefully and washed twice with isotonic phosphate bufferedsaline (PBS) by centrifugation at 250×g for 10 minutes. The resultingcell pellet was then resuspended in RPMI 1640 medium containingL-glutamine (MediaTech, Inc., Herndon, Va.) and supplemented with 10%heat inactivated FCS and penicillin-streptomycin (100 U/ml). Cells werecultured in 24 well plates for different time periods at 1×10⁶cells/ml/well in the presence or absence of oligonucleotides. At the endof the incubation period, supernatants were harvested and stored frozenat −70° C. until assayed for various cytokines including IL-6 (BDPharmingen, San Diego, Calif.), IL-10 (BD Pharmingen), IL-12 (BioSourceInternational, Camarillo, Calif.), IFN-α (BioSource International) and−γ (BD Pharmingen) and TNF-α (BD Pharmingen) by sandwich ELISA. Theresults are shown in Table 5 below.

In all instances, the levels of IL-12 and IL-6 in the cell culturesupernatants were calculated from the standard curve constructed underthe same experimental conditions for IL-12 and IL-6, respectively. Thelevels of IL-10, IFN-gamma and TNF-α in the cell culture supernatantswere calculated from the standard curve constructed under the sameexperimental conditions for IL-10, IFN-gamma and TNF-α, respectively.

TABLE 5 Immunomer Structure and Immunostimulatory Activity in Human PBMCCultures Oligo Oligo Length/ IL-12(pg/mL) IL-6(pg/mL) IL-10(pg/mL)IFN-γ(pg/mL) TNF-α(pg/mL) No. Sequences and Modification (5′-3′) or EachChain D1 D2 D1 D2 D1 D2 D1 D2 D1 D2 25 5′-CTATCTGTCGTTCTCTGT-3′18mer(PS) 184 332 3077 5369 37  88 125 84 537 nt (SEQ ID NO: 17)26(SEQIDNO:18)

11mer(PS) 237 352 3724 4892 48 139 251 40 681 nt D1 and D2 are donors 1and 2.

TABLE 5A Immunomer Structure and Immunostimulatory Activity in BALB/cMouse Spleen Cell Cultures Oligo Oligo Length/ IL-12(pg/mL) IL-6(pg/mL)No Sequences and Modification (5′-3′) or Each Chain 3 □g/mL 10 □g/mL26(SEQIDNO:18)

11mer (PS) 870 10670 27(SEQIDNO:19)

11mer (PS) 1441 7664 28(SEQIDNO:20)

11mer (PS) 1208 1021 29(SEQIDNO:21)

11mer (PS) 162 1013 30(SEQIDNO:22)

14mer (PO) 264 251 31(SEQIDNO:23)

14mer (PO) 149 119 32(SEQIDNO:24)

11mer (PS) 2520 9699 33(SEQIDNO:25)

11mer (PS) 2214 16881 34(SEQIDNO:26)

11mer (PS) 3945 10766 35(SEQIDNO:27)

11mer (PS) 2573 19411 36(SEQIDNO:28)

14mer (PO) 2699 408 37(SEQIDNO:29)

14mer (PO) 839 85 38(SEQIDNO:30)

14mer (PO) 143 160Normal phase represents a phosphorothioate linkage; Italic phaserepresents a phosphodiester linkage.

In addition, the results shown in FIGS. 7A-C demonstrate thatOligonucleotide 2, with two accessible 5′ ends elevates IL-12 and IL-6,but not IL-10 at lower concentrations than Oligonucleotides 1 or 3, withone or zero accessible 5′ ends, respectively.

Example 5 Effect of Chain Length on Immunostimulatory Activity ofImmunomers

In order to study the effect of length of the oligonucleotide chains,immunomers containing 18, 14, 11, and 8 nucleotides in each chain weresynthesized and tested for immunostimulatory activity, as measured bytheir ability to induce secretion of the cytokines IL-12 and IL-6 inBALB/c mouse spleen cell cultures (Tables 6-8). In this, and allsubsequent examples, cytokine assays were carried out in BALB/c spleencell cultures as described in Example 4.

TABLE 6 Immunomer Structure and Immunostiniulatory Activity OligoLength/ IL-12(pg/mL) IL-8(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain @ 0.3 μg/mL @ 0.3 μg/mL  4(SEQIDNO:2)5′-CTATCTGACGTTCTCTGT-3′ 18mer 1802 176 39(SEQIDNO:2)

18mer 1221 148 40(SEQIDNO:8)

14mer 2107 548 41(SEQIDNO:13)

11mer 3838 1191 42

 8mer 567 52

TABLE 7 Immunomer Structure and Immunostimulatory Activity Oligo Length/IL-12(pg/mL) IL-6(pg/mL) No. Sequences and Modification (5′-3′) or EachChain 1 μg/mL 1 μg/mL 25(SEQIDNO:17) 5′-CTATCTGTCGTTCTCTGT-3′ 18mer 29165 43(SEQIDNO:17)

18mer 430 39 44(SEQIDNO:31)

14mer 813 59 45(SEQIDNO:32)

12mer 1533 123 46(SEQIDNO:33)

11mer 2933 505 47

 8mer 1086 26 48

 7mer 585 34 49

 6mer 764 76 50

5mer 28 29

TABLE 8 Immunomer Structure and Immunostimulatory Activity Oligo Length/IL-12(pg/mL) IL-6(pg/mL) No. Sequences and Modification (5′-3′) or EachChain 1 μg/mL 1 μg/mL 51(SEQIDNO:34) 5′-CTCACTTTCGTTCTCTGT-3′ 18mer 9173 52(SEQIDNO:34)

18mer 502 99 53(SEQIDNO:35)

14mer 683 119 54(SEQIDNO:36)

12mer 633 102 55

 8mer 687 243 56

 7mer 592 1252

The results suggest that the immunostimulatory activity of immunomersincreased as the length of the oligonucleotide chains is decreased from18-mers to 7-mers. Immunomers having oligonucleotide chain lengths asshort as 6-mers or 5-mers showed immunostimulatory activity comparableto that of the 18-mer oligonucleotide with a single 5′ end. However,immunomers having oligonucleotide chain lengths as short as 6-mers or5-mers have increased immunostimulatory activity when the linker is inthe length of from about 2 angstroms to about 200 angstroms.

Example 6 Immunostimulatory Activity of Immunomers Containing ANon-Natural Pyrimidine or Non-Natural Purine Nucleoside

As shown in Tables 9-11, immunostimulatory activity was maintained forimmunomers of various lengths having a non-natural pyrimidine nucleosideor non-natural purine nucleoside in the immunostimulatory dinucleotidemotif.

TABLE 9 Immunomer Structure and Immunostiniulatory Activity OligoLength/ IL-12(pg/mL) IL-8(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain @ 3 μg/mL @ 3 μg/mL 51(SEQIDNO:34)5′-CTCACTTTCGTTCTCTGT-3′ 18mer 1404 348 57(SEQIDNO:37)

11mer 591 365 58(SEQIDNO:38)

11mer 303 283 59

 8mer 55 66 60

 8mer 242 143

TABLE 10 Immunomer Structure and Immunostiniulatory Activity OligoLength/ IL-12(pg/mL) IL-8(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain @ 3 μg/mL @ 3 μg/mL 25(SEQIDNO:17)5′-CTATCTGTCGTTCTCTGT-3′ 18mer 379 339 61(SEQIDNO:39)

11mer 1127 470 62(SEQIDNO:18)

11mer 787 296 63

 8mer 64 126 64

 8mer 246 113

TABLE 11 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12(pg/mL) IL-6(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain 3 μg/mL 3 μg/mL  4(SEQIDNO:2) 5′-CTATCTGACGTTCTCTGT-3′18mer 1176 1892 65(SEQIDNO:4)

18mer 443 192 66(SEQIDNO:5)

18mer 627 464 67(SEQIDNO:6)

14mer 548 152 68(SEQIDNO:7)

14mer 1052 1020  69(SEQIDNO:40)

11mer 2050 2724  70(SEQIDNO:24)

11mer 1780 1741 71

 8mer 189 55 72

 8mer 397 212

Example 7 Effect of the Linker on Immunostimulatory Activity

In order to examine the effect of the length of the linker connectingthe two oligonucleotides, immunomers that contained the sameoligonucleotides, but different linkers were synthesized and tested forimmunostimulatory activity. The results shown in Table 12 suggest thatlinker length plays a role in the immunostimulatory activity ofimmunomers. The best immunostimulatory effect was achieved with C3- toC6-alkyl linkers or abasic linkers having interspersed phosphatecharges.

TABLE 12 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12(pg/mL) IL-6(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain 0.3 μg/mL 1 μg/mL 4(SEQIDNO:2) 5′-CTATCTGACGTTCTCTGT-3′18mer 257 635 73(SEQIDNO:41)

10mer 697 1454 74(SEQIDNO:41)

10mer 1162 669 75(SEQIDNO:41)

10mer 1074 1375 76(SEQIDNO:41)

10mer 563 705 77(SEQIDNO:41)

10mer 264 543 78(SEQIDNO:41)

10mer 1750 2258 79(SEQIDNO:41)

10mer 2255 2034 80(SEQIDNO:41)

10mer 1493 1197 81(SEQIDNO:41)

10mer 3625 2642 82(SEQIDNO:41)

10mer 4248 2988 83(SEQIDNO:41)

10mer 1241 1964

Example 8 Effect of Oligonucleotide Backbone on ImmunostimulatoryActivity

In general, immunostimulatory oligonucleotides that contain naturalphosphodiester backbones are less immunostimulatory than are the samelength oligonucleotides with a phosphorothioate backbones. This lowerdegree of immunostimulatory activity could be due in part to the rapiddegradation of phosphodiester oligonucleotides under experimentalconditions. Degradation of oligonucleotides is primarily the result of3′-exonucleases, which digest the oligonucleotides from the 3′ end. Theimmunomers of this example do not contain a free 3′ end. Thus,immunomers with phosphodiester backbones should have a longer half lifeunder experimental conditions than the corresponding monomericoligonucleotides, and should therefore exhibit improvedimmunostimulatory activity. The results presented in Table 13demonstrate this effect, with Immunomers 84 and 85 exhibitingimmunostimulatory activity as determined by cytokine induction in BALB/cmouse spleen cell cultures.

TABLE 13 Immunomer Structure and Immunostimulatory Activity OligoLength/ IL-12(pg/mL) IL-6(pg/mL) No. Sequences and Modification (5′-3′)or Each Chain 0.3 μg/mL 1 μg/mL  4(SEQIDNO:2) 5′-CTATCTGACGTTCTCTGT-3′18mer 225 1462 84(SEQIDNO:8)

14mer 1551 159 85(SEQIDNO:8)

14mer 466 467 L = C3-Linker

Example 9 Synthesis of Immunomers 73-92

Oligonucleotides were synthesized on 1 μmol scale using an automated DNAsynthesizer (Expedite 8909 PerSeptive Biosystems). Deoxynucleosidephosphoramidites were obtained from Applied Biosystems (Foster City,Calif.). 7-Deaza-2′-deoxyguanosine phosphoramidite was obtained fromGlen Research (Sterling Va.). 1,3-Bis-DMT-glycerol-CPG was obtained fromChemGenes (Ashland, Mass.). Modified nucleosides were incorporated intothe oligonucleotides at specific site using normal coupling cycles.After the synthesis, oligonucleotides were deprotected usingconcentrated ammonium hydroxide and purified by reversed-phase HPLC,followed by dialysis. Purified oligonucleotides as sodium salt form werelyophilized prior to use. Purity of oligonucleotides was checked by CGEand MALDI-TOF MS (Bruker Proflex III MALDI-TOF Mass spectrometer).

Example 11 Immunomer Stability

Oligonucleotides were incubated in PBS containing 10% bovine serum at37° C. for 4, 24 or 48 hours. Intact oligonucleotide was determined bycapillary gel electrophoresis. The results are shown in Table 14.

TABLE 14 Digestion of Oligonucleotides in 10% Bovine Serum PBS SolutionCE analysis of oligos (% intact Oligo Sequences and Modification oligoremained after digestion) No. (5′-3+) after 4 h After 24 h after 48 h  45-CTATCTGACGTTCTCTGT- 90.9 71.8 54.7 (SEQ 3′/PS ID NO: 2) 26(5′-TCTGTCGTTCT)₂S/PS 97.1 91.0 88.1 (SEQ (G = dG^(deaza)) ID NO: 42) 86(5′-CTGTCGTTCTCTGT)₂S/PO 37.8 22.5 (SEQ ID NO: 43) 87 (5′- 73.1 56.836.8 (SEQ XXCTGTCGTTCTCTGT)₂S/PO ID NO: 31) 88 (5′-UCTGTCGTTCTCTGT)₂S/PO48.4 36.7 (SEQ ID NO: 44) X = C3-Linker, U, C = 2′-OMe-ribonucleoside

Example 12 Effect of Accessible 5′ Ends on Immunostimulatory Activity

BALB/c mouse (4-8 weeks) spleen cells were cultured in RPMI completemedium. Murine macrophage-like cells, J774 (American Type CultureCollection, Manassas, VA) were cultured in Dulbecco's modified Eagle'smedium supplemented with 10% (v/v) FCS and antibiotics (100 IU/mL ofpenicillin G/streptomycin). All other culture reagents were purchasedfrom Mediatech (Gaithersburg, MD).

ELISAs for IL-12 and IL-6. BALB/c mouse spleen or J774 cells were platedin 24-well dishes at a density of 5×10⁶ or 1×10⁶ cells/mL, respectively.The CpG DNA dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)was added to a final concentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0μg/mL to mouse spleen cell cultures and 1.0, 3.0, or 10.0 μg/mL to J774cell cultures. The cells were then incubated at 37° C. for 24 hr and thesupernatants were collected for ELISA assays. The experiments wereperformed two or three times for each CpG DNA in triplicate for eachconcentration.

The secretion of IL-12 and IL-6 was measured by sandwich ELISA. Therequired reagents, including cytokine antibodies and standards werepurchased from PharMingen. ELISA plates (Costar) were incubated withappropriate antibodies at 5 μg/mL in PBSN buffer (PBS/0.05% sodiumazide, pH 9.6) overnight at 4° C. and then blocked with PBS/1% BSA at37° C. for 30 min. Cell culture supernatants and cytokine standards wereappropriately diluted with PBS/1% BSA, added to the plates intriplicate, and incubated at 25° C. for 2 hr. Plates were washed andincubated with 1 μg/mL of appropriate biotinylated antibody andincubated at 25° C. for 1.5 hr. The plates were washed extensively withPBS/0.05% Tween 20 and then further incubated at 25° C. for 1.5 hr afterthe addition of streptavidine-conjugated peroxidase (Sigma). The plateswere developed with Sure Blue™ (Kirkegaard and Perry) chromogenicreagent and the reaction was terminated by adding Stop Solution(Kirkegaard and Perry). The color change was measured on a Ceres 900 HDISpectrophotometer (Bio-Tek Instruments) at 450 nm. The levels of IL-12and IL-6 in the cell culture supernatants were calculated from thestandard curve constructed under the same experimental conditions forIL-12 and IL-6, respectively.

The results are shown in Table 15.

TABLE 15 Phosphorothioate CpG DNA sequences and modifications used inthe study^(a) (all sequences represent SEQ ID NO: 45) CpG DNA # SequenceLength 5′-end 3′-end 89 5′-TCCATGACGTTCCTGATGC-3′ 19-mer 1 1 905′-TCCATGACGTTCCTGATGC-3′-b 19-mer 1 blocked 915′-TCCATGACGTTCCTGATGC-3′-3′-g-5′ 20-mer 2 blocked 925′-TCCATGACGTTCCTGATGC-3′-3′-h-5′ 23-mer 2 blocked 935′-TCCATGACGTTCCTGATGC-3′-3′-i-5′ 27-mer 2 blocked 945′-TCCATGACGTTCCTGATGC-3′-3′-j-5′ 38-mer 2 blocked 95b-5′-TCCATGACGTTCCTGATGC-3′ 19-mer blocked 1 963′-c-5′-5′-TCCATGACGTTCCTGATGC-3′ 20-mer blocked 2 973′-d-5′-5′-TCCATGACGTTCCTGATGC-3′ 23-mer blocked 2 983′-e-5′-5′-TCCATGACGTTCCTGATGC-3′ 27-mer blocked 2 993′-f-5′-5′-TCCATGACGTTCCTGATGC-3′ 38-mer blocked 2 1005′-TCCATGACGTTCCTGATGC-3′-k 19-mer 1 blocked 1011-5′-TCCATGACGTTCCTGATGC-3′ 19-mer blocked 1 ^(a)See Chart I forchemical structures b-1; 5′-CG-3′ dinucleotide is shown underlined.Chart 1 (SEQ ID NOS 46, 47 and 48, respectively in order from top tobottom)

1: R, R′= a —H (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k): X = CH₂OH; (l): X = H.

TABLE 16 Induction of IL-12 and IL-6 secretion by CpG DNA-conjugates inBALB/c mice spleen cell cultures IL-6 (pg/mL) ± SD CpG IL-12 (pg/mL) ±SD 10.0 DNA #^(a) 0.1 μg/mL 0.3 μg/mL 1.0 μg/mL 3.0 μg/mL 10.0 μg/mL 0.1μg/mL 0.3 μg/mL 1.0 μg/mL 3.0 μg/mL μg/mL 89  991 ± 121 1820 ± 224 2391± 175 3507 ± 127 2615 ± 279  652 ± 48 2858 ± 180 13320 ± 960 18625 ±1504 17229 ± 1750 90  526 ± 32 2100 ± 175 1499 ± 191 3019 ± 35 3489 ±162 1387 ± 152 1426 ± 124  5420 ± 370 19096 ± 484 19381 ± 2313 91 1030 ±11 1348 ± 102 2060 ± 130 3330 ± 130 3582 ± 259  923 ± 22 2542 ± 81  9054± 120 14114 ± 179 13693 ± 264 92 1119 ± 159 1726 ± 207 2434 ± 100 2966 ±204 3215 ± 464  870 ± 146 1905 ± 56  7841 ± 350 17146 ± 1246 15713 ± 69393 1175 ± 68 2246 ± 124 1812 ± 75 2388 ± 320 2545 ± 202 1152 ± 238 3499± 116  7142 ± 467 14064 ± 167 13566 ± 477 94 1087 ± 121 1705 ± 163 1797± 141 2522 ± 195 3054 ± 103 1039 ± 105 2043 ± 157  4848 ± 288 15527 ±224 21021 ± 1427 95 1173 ± 107 2170 ± 155 2132 ± 58 2812 ± 203 3689 ± 94 807 ± 0.5  927 ± 0.5  3344 ± 0.5 10233 ± 0.5  9213 ± 0.5 96  866 ± 511564 ± 63 1525 ± 63 2666 ± 97 4030 ± 165  750 ± 63 1643 ± 30  5559 ± 41511549 ± 251 11060 ± 651 97  227 ± 3  495 ± 96 1007 ± 68  897 ± 15 1355 ±97  302 ± 18  374 ± 22  1000 ± 68  9106 ± 271 13077 ± 381 98  139 ± 18 211 ± 12  452 ± 22  458 ± 29 1178 ± 237  220 ± 23  235 ± 18  383 ± 35 1706 ± 33 11530 ± 254 99  181 ± 85  282 ± 105  846 ± 165 2082 ± 1853185 ± 63  467 ± 122  437 ± 85  1697 ± 283  9781 ± 13 11213 ± 294 Medium 86 ± 6  60 ± 12 ^(a)See Table 1 for sequences.

Taken together, the current results suggest that an accessible 5′-end ofCpG DNA is required for its optimal immunostimulatory activity andsmaller groups such as a phosphorothioate, a mononucleotide, or adinucleotide do not effectively block the accessibility of the 5′-end ofCpG DNA to receptors or factors involved in the immunostimulatorypathway. However, the conjugation of molecules as large as fluoresceinor larger at the 5′-end of CpG DNA could abrogate immunostimulatoryactivity. These results have a direct impact on the studies ofimmunostimulatory activity of CpG DNA-antigen/vaccine/monoclonalantibody (mAb) conjugates. The conjugation of large molecules such asvaccines or mAbs at the 5′-end of a CpG DNA could lead to suboptimalimmunostimulatory activity of CpG DNA. The conjugation of functionalligands at the 3′-end of CpG DNA not only contributes to increasednuclease stability but also increased immunostimulatory potency of CpGDNA in vivo.

Example 13 Effect of Linkers on Cytokine Secretion

The following oligonucleotides were synthesized for this study. Each ofthese modified oligonucleotides can be incorporated into an immunomer.

TABLE 17 Sequences of CpG DNA showing the position of substitution. CpGDNA Number Sequence (5′--->3′)^(a) 102 CCTACTAGCGTTCTCATC (SEQ ID NO:49) 103 CCTACTAGC2TTCTCATC (N/A) 104 CCTACT2GCGTTCTATC (SEQ ID NO: 50)105 CCTA2TAGCGTTCTCATC (SEQ ID NO: 51) 106 CCT22TAGCGTTCTCATC (SEQ IDNO: 51) 107 22TACTAGCGTTCTCATC (SEQ ID NO: 52) 108 CCTACTAGCGT2CTCATC(SEQ ID NO: 53) 109 CCTACTAGCGTTC2CATC (SEQ ID NO: 54) 110CCTACTAGCGTTC22ATC (SEQ ID NO: 54) 111 CCT6CTAGCGTTCTCATC (SEQ ID NO:55) 112 CCTACTAGCGTTC6CATC (SEQ ID NO: 54) 113 CCT7CTAGCGTTCTCATC (SEQID NO: 55) 114 CCTACTAGCGTTC7CATC (SEQ ID NO: 54) 4 CTATCTGACGTTCTCTGT(SEQ ID NO: 2) 115 CTAT1TGACGTTCTCTGT (SEQ ID NO: 3) 116CTA1CTGACGTTCTCTGT (SEQ ID NO: 8) 117 CTATCTG2CGTTCTCTGT (SEQ ID NO: 56)118 CTATC2GACGTTCTCTGT (SEQ ID NO: 57) 119 CTA2CTGACGTTCTCTGT (SEQ IDNO: 8) 120 22222TGACGTTCTCTGT (SEQ ID NO: 3) 121 z2222TGACGTTCTCTGT (SEQID NO: 3) 122 zz222TGACGTTCTCTGT (SEQ ID NO: 3) 123 zzz22TGACGTTCTCTGT(SEQ ID NO: 3) 124 zzzz2TGACGTTCTCTGT (SEQ ID NO: 3) 125CTAT3TGACGTTCTCTGT (SEQ ID NO: 3) 126 CTA3CTGACGTTCTCTGT (SEQ ID NO: 8)127 CTA33TGACGTTCTCTGT (SEQ ID NO: 3) 128 zzz33TGACGTTCTCTGT (SEQ ID NO:3) 129 CTAT4TGACGTTCTCTGT (SEQ ID NO: 3) 130 CTA4CTGACGTTCTCTGT (SEQ IDNO: 8) 131 CTA44TGACGTTCTCTGT (SEQ ID NO: 3) 132 zzz44TGACGTTCTCTGT (SEQID NO: 3) 133 CTAT5TGACGTTCTCTGT (SEQ ID NO: 3) 134 CTA5CTGACGTTCTCTGT(SEQ ID NO: 8) 135 CTA55TGACGTTCTCTGT (SEQ ID NO: 3) 136zzz55TGACGTTCTCTGT (SEQ ID NO: 3) 137 CTA6CTGACGTTCTCTGT (SEQ ID NO: 8)138 CTATCTGACGTTC6CTGT (SEQ ID NO: 58) 139 CTA7CTGACGTTCTCTGT (SEQ IDNO: 8) 140 CTATCTGACGTTC7CTGT (SEQ ID NO: 58) 141 CTATCTG8CGTTCTCTGT(SEQ ID NO: 59 142 CTATCT8ACGTTCTCTGT (SEQ ID NO: 60) 143CTATC8GACGTTCTCTGT (SEQ ID NO: 57) 144 CTAT8TGACGTTCTCTGT (SEQ ID NO: 3)145 CTA8CTGACGTTCTCTGT (SEQ ID NO: 8) 146 CTATCTGACG8TCTCTGT (SEQ ID NO:61) 147 CTATCTGACGT8CTCTGT (SEQ ID NO: 62) 148 CTATCTGACGTT8TCTGT (SEQID NO: 63) 149 CTATCTGACGTTC8CTGT (SEQ ID NO: 58) 150 CTATCTG9CGTTCTCTGT(SEQ ID NO: 59) 151 CTATCT9ACGTTCTCTGT (SEQ ID NO: 60) 152CTA9CTGACGTTCTCTGT (SEQ ID NO: 8) 153 CTATCTGACGT9CTCTGT (SEQ ID NO: 62)154 CTATCTGACGTTC9CTGT (SEQ ID NO: 58) ^(a)See FIG. 14 for the chemicalstructures of substitutions 1-9. All CpG DNAs are phosphorothioatebackbone modified.

To evaluate the optimal linker size for potentiation ofimmunostimulatory activity, we measured IL-12 and IL-6 secretion inducedby modified CpG DNAs in BALB/c mouse spleen cell cultures. All CpG DNAsinduced concentration-dependent IL-12 and IL-6 secretion. FIG. 15 showsdata obtained at 1 μg/mL concentration of selected CpG DNAs, 116, 119,126, 130, and 134, which had a linker at the fifth nucleotide positionin the 5′-flanking sequence to the CpG dinucleotide compared with theparent CpG DNA. The CpG DNAs, which contained C2-(1), C3-(2), andC4-linkers (3), induced secretion of IL-12 production similar to that ofthe parent CpG DNA 4. The CpG DNA that contained C6 and C9-linkers (4and 5) at the fifth nucleotide position from CpG dinucleotide in the5′-flanking sequence induced lower levels of IL-12 secretion than didthe parent CpG DNA (FIG. 15), suggesting that substitution of linkerslonger than a C4-linker results in the induction of lower levels ofIL-12. All five CpG DNAs, which had linkers, induced two to three timeshigher IL-6 secretion than did the parent CpG DNA. The presence of alinker in these CpG DNAs showed a significant effect on the induction ofIL-6 compared with CpG DNAs that did not have a linker. However, we didnot observe length-dependent linker effect on IL-6 secretion.

To examine the effect on immunostimulatory activity of CpG DNAcontaining ethylenegylcol-linkers, we synthesized CpG DNAs 137 and 138,in which a triethyleneglycol-linker (6) is incorporated at the fifthnucleotide position in the 5′- and at the fourth nucleotide position inthe 3′-flanking sequences to the CpG dinucleotide, respectively.Similarly, CpG DNAs 139 and 140 contained a hexaethyleneglycol-linker(7) in the 5′- or the 3′-flanking sequence to the CpG dinucleotide,respectively. All four modified CpG DNAs (137-140) were tested in BALB/cmouse spleen cell cultures for cytokine induction (IL-12, IL-6, andIL-10) in comparison with parent CpG DNA 4. All CpG DNAs inducedconcentration-dependent cytokine production over the concentration rangetested (0.03-10.0 μg/mL) (data not shown). The levels of cytokinesinduced at 0.3 μg/mL concentration of CpG DNAs 137-140 are shown inTable 18. CpG DNAs 137 and 139, which had an ethyleneglycol-linker inthe 5′-flanking sequence induced higher levels of IL-12 (2106±143 and2066±153 pg/mL) and IL-6 (2362±166 and 2507±66 pg/mL) secretion than didparent CpG DNA 4 (Table 18). At the same concentration, 137 and 139induced slightly lower levels of IL-10 secretion than did the parent CpGDNA (Table 18). CpG DNA 138, which had a shorter ethyleneglycol-linker(6) in the 3′-flanking sequence induced IL-12 secretion similar to thatof the parent CpG DNA, but significantly lower levels of IL-6 and IL-10(Table 18). CpG DNA 140, which had a longer ethyleneglycol-linker (7)induced significantly lower levels of all three cytokines testedcompared with the parent CpG DNA (Table 18).

Though triethyleneglycol-linker (6) had a chain length similar to thatof C9-linker (5), the CpG DNA containing triethyleneglycol-linker hadbetter immunostimulatory activity than did CpG DNA containing C9-linker,as determined by induction of cytokine secretion in spleen cellcultures. These results suggest that the lower immunostimulatoryactivity observed with CpG DNA containing longer alkyl-linkers (4 and 5)may not be related to their increased length but to their hydrophobiccharacteristics. This observation prompted us to examine substitution ofbranched alkyl-linkers containing hydrophilic functional groups onimmunostimulatory activity.

TABLE 18 Cytokine secretion induced by CpG DNAs containing anethyleneglycol- linker in BALB/c mice spleen cell cultures. CpG DNACytokine, pg/mL Number IL-12 IL-6 IL-10  4 1887 ± 233 2130 ± 221 86 ± 14137 2106 ± 143 2362 ± 166 78 ± 21 138 1888 ± 259 1082 ± 25  47 ± 14 1392066 ± 153 2507 ± 66  73 ± 17 140 1318 ± 162 476 ± 13 25 ± 5  Medium  84± 13 33 ± 6 2 ± 1

To test the effect on immunostimulatory activity of CpG DNA containingbranched alkyl-linkers, two branched alkyl-linkers containing a hydroxyl(8) or an amine (9) functional group were incorporated in parent CpG DNA4 and the effects on immunostimulatory activity of the resultingmodified CpG DNAs (150-154-Table 19) were examined. The data obtainedwith CpG DNAs 150-154, containing amino-linker 9 at different nucleotidepositions, in BALB/c mouse spleen cell cultures (proliferation) and invivo (splenomegaly) are shown in Table 19.

TABLE 19 Spleen cell proliferation induced by CpG DNA containing anaminobutyryl propanediol-linker in BALB/c mice spleen cell cultures andsplenomegaly in BALB/c mice. CpG Spleen cell Spleen DNA proliferationweight Number^(a) (PI)^(b) (mg)^(c)  4 3.7 ± 0.8 121 ± 16 150 2.5 ± 0.6107 ± 11 151 9.2 ± 0.7 169 ± 16 152 8.8 ± 0.4 220 ± 8  153 7.6 ± 0.7 127± 24 154  7.8 ± 0.04 177 ± 12 M/V 1.2 ± 0.3 102 ± 8  LPS 2.8 ± 0.5 ND

Parent CpG DNA 4 showed a proliferation index of 3.7±0.8 at aconcentration of 0.1 μg/mL. At the same concentration, modified CpG DNAs151-154 containing amino-linker 9 at different positions caused higherspleen cell proliferation than did the parent CpG DNA (Table 19). Asobserved with other linkers, when the substitution was placed adjacentto CpG dinucleotide (150), a lower proliferation index was notedcompared with parent CpG DNA (Table 19), further confirming that theplacement of a linker substitution adjacent to CpG dinucleotide has adetrimental effect on immunostimulatory activity. In general,substitution of an amino-linker for 2′-deoxyribonucleoside in the5′-flanking sequence (151 and 152) resulted in higher spleen cellproliferation than found with the substitution in the 3′-flankingsequence (153 and 154). Similar results were observed in thesplenomegaly assay (Table 19), confirming the results observed in spleencell cultures. Modified CpG DNAs containing glycerol-linker (8) showedimmunostimulatory activity similar to or slightly higher that thatobserved with modified CpG DNA containing amino-linker (9) (data notshown).

In order to compare the immunostimulatory effects of CpG DNA containinglinkers 8 and 9, we selected CpG DNAs 145 and 152, which hadsubstitution in the 5′-flanking sequence and assayed their ability toinduce cytokines IL-12 and IL-6 secretion in BALB/c mouse spleen cellcultures. Both CpG DNAs 145 and 152 induced concentration-dependentcytokine secretion. FIG. 4 shows the levels of IL-12 and IL-6 induced by145 and 152 in mouse spleen cell cultures at 0.3 μg/mL concentrationcompared with parent CpG DNA 4. Both CpG DNAs induced higher levels ofIL-12 and IL-6 than did parent CpG DNA 4. CpG DNA containingglycerol-linker (8) induced slightly higher levels of cytokines(especially IL-12) than did CpG DNA containing amino-linker (9) (FIG.16). These results further confirm that the linkers containinghydrophilic groups are more favorable for immunostimulatory activity ofCpG DNA.

We examined two different aspects of multiple linker substitutions inCpG DNA. In one set of experiments, we kept the length of nucleotidesequence to 13-mer and incorporated one to five C3-linker (2)substitutions at the 5′-end (120-124). These modified CpG DNAs permittedus to study the effect of an increase in the length of linkers withoutcausing solubility problems. In the second set of experiments, weincorporated two of the same linker substitutions (3, 4, or 5) inadjacent positions in the 5′-flanking sequence to the CpG dinucleotideto study if there would be any additive effect on immunostimulatoryactivity.

Modified CpG DNAs were studied for their ability to induce cytokineproduction in BALB/c mouse spleen cell cultures in comparison withparent CpG DNA 4. All CpG DNAs induced concentration-dependent cytokineproduction. The data obtained at 1.0 μg/mL concentration of CpG DNAs isshown in Table 20. In this assay, parent CpG DNA 4 induced 967±28 pg/mLof IL-12, 1593±94 pg/mL of IL-6, and 14±6 pg/mL of IL-10 secretion at 1μg/mL of concentration. The data presented in Table 20 suggest that asthe number of linker substitutions decreased IL-12 induction decreased.However, the induction of lower levels of IL-12 secretion by CpG DNAs123 and 124 could be the result of the shorter length of CpG DNAs. Ourstudies with unmodified CpG DNA shorter than 15-nucleotides showedinsignificant immunostimulatory activity (data not shown). Neitherlength nor the number of linker substitutions have a lesser effect onIL-6 secretion. Though IL-10 secretion increased with linkersubstitutions, the overall IL-10 secretion by these CpG DNAs wasminimal.

CpG DNAs containing two linker substitutions (linker 3-127;linker-4-131; linker-5-135) at the fourth and fifth positions in the5′-flanking sequences to the CpG dinucleotide and the corresponding5′-truncated versions 128, 132, and 136, respectively, were tested fortheir ability to induce cytokine secretion in BALB/c mouse spleen cellcultures. The levels of IL-12 and IL-6 secreted at 1.0 μg/mLconcentration are shown in FIG. 17. The results presented in FIG. 17suggest that the immunostimulatory activity is dependent on the natureof the linker incorporated. The substitution of the fourth and fifthnucleosides with C4-linker 3 (CpG DNA 127) had an insignificant effecton cytokine secretion compared with parent CpG DNA 4, suggesting thatthe nucleobase and sugar ring at these positions are not required forreceptor recognition and/or binding. The deletion of the nucleotidesbeyond the linker substitutions (CpG DNA 128) caused higher IL-12 andIL-6 secretion than that found with CpG DNAs 4 and 127. As expected, thesubstitution of two C6-linkers (4) resulted in IL-12 secretion lowerthan and IL-6 secretion similar to that induced by parent CpG DNA 4. The5′-truncated CpG DNA 132 induced higher cytokine secretion than did CpGDNA 131. The CpG DNAs 135 and 136, which had two C9-linkers (5), inducedinsignificant cytokine secretion, confirming the results obtained withmono-substituted CpG DNA containing the same linker as described above.

Example 14 Effect of Phosphodiester Linkages on Cytokine Induction

To test the effect of phosphodiester linkages on immunomer-inducedcytokine induction, the following molecules were synthesized.

TABLE 21 PO-Immunomer sequences and analytical data (SEQ ID NOS 2 and64, respectively in order of apperance CpG Molecular Weight DNASequence^(a) Backbone^(b) Calculated Found^(c) 4

PS 5702 5704 155

PO 5432 5428 156

PO 8656 8649 157

PO 9208 9214

^(a)Arrows indicate 5′-3′ directionality of CpG dinucleotide in each DNAmolecule and structures of X and V are shown in boxes. ^(b)PS and POstand for phosphorothioate and phosphodiester backbones, respectively.^(c)As determined by MALDI-TOF mass spectrometry.

PS-CpG DNA 4 (Table 21) was found to induce an immune response in mice(data not shown) with PO-CpG DNA 155 serving as a control. PO-immunomers156 and 157 each contain two identical, truncated copies of the parentCpG DNA 155 joined through their 3′-ends via a glyceryl linker, X (Table21). While 156 and 157 each contain the same oligonucleotide segments of14 bases, the 5′-ends of 157 were modified by the addition of twoC3-linkers, Y (Table 21). All oligonucleotides 4, 155-157 contain a‘GACGTT’ hexameric motif known to activate the mouse immune system.

The stability of PO-immunomers against nucleases was assessed byincubating CpG DNAs 4, 155-157 in cell culture medium containing 10%fetal bovine serum (FBS) (non-heat-inactivated) at 37° C. for 4, 24, and48 hr. Intact CpG DNA remaining in the reaction mixtures were thendetermined by CGE. FIGS. 18 A-D shows the nuclease digestion profiles ofCpG DNAs 4, 155-157 incubated in 10% FBS for 24 hr. The amount offull-length CpG DNA remaining at each time point is shown in FIG. 18 E.As expected, the parent PS-CpG DNA 4 is the most resistant to serumnucleases. About 55% of 18-mer 4 remained undegraded after 48 hrincubation. In contrast, only about 5% of full-length PO-immunomer 155remained after 4 hr under the same experimental conditions confirmingthat DNA containing phosphodiester linkages undergoes rapid degradation.As expected, both PO-immunomers 156 and 157 were more resistant than 155to serum nucleases. After 4 hr, about 62% and 73% of 156 and 157respectively were intact compared with about 5% of 155 (FIG. 18 E). Evenafter 48 hr, about 23% and 37% of 156 and 157, respectively, remainedundegraded. As well as showing that 3′-3′-linked PO-immunomers are morestable against serum nucleases, these studies indicate that chemicalmodifications at the 5′-end can further increase nuclease stability.

The immunostimulatory activity of CpG DNAs was studied in BALB/c andC3H/HeJ mice spleen cell cultures by measuring levels of cytokines IL-12and IL-6 secreted. All CpG DNAs induced a concentration-dependentcytokine secretion in BALB/c mouse spleen cell cultures (FIG. 19). At 3μg/mL, PS-CpG DNA 4 induced 2656±256 and 12234±1180 pg/mL of IL-12 andIL-6 respectively. The parent PO-CpG DNA 155 did not raise cytokinelevels above background except at a concentration of 10 μg/mL. Thisobservation is consistent with the nuclease stability assay results. Incontrast, PO-immunomers 156 and 157 induced both IL-12 and IL-6secretion in BALB/c mouse spleen cell cultures.

The results presented in FIG. 19 show a clear distinction in cytokineinduction profiles of PS- and PO-CpG DNAs. PO-immunomers 156 and 157induced higher levels of IL-12 than did PS-CpG DNA 4 in BALB/c mousespleen cell cultures (FIG. 19A). In contrast, at concentrations up to 3μg/mL, they produced negligible amounts of IL-6 (FIG. 19B). Even at thehighest concentration (10 μg/mL), PO-immunomer 156 induced significantlyless IL-6 than did PS-CpG DNA 4. The presence of C3 linkers at the5′-terminus of PO-immunomer 157 resulted in slightly higher levels ofIL-6 secretion compared with 156. However, importantly, the levels ofIL-6 produced by PO-immunomer 157 are much lower than those induced byPS CpG DNA 4. The inset of FIG. 19A shows the ratio of IL-12 to IL-6secreted at 3 μg/mL concentration. In addition to increasing IL-12secretion, PO-immunomers 156 and 157 induced higher levels of IFN-γ thandid PS-CpG DNA 4 in BALB/c mouse spleen cell cultures (data not shown).

The different cytokine profiles induced by PO- and PS-CpG DNAs in BALB/cmouse spleen cell cultures prompted us to study the pattern of cytokineinduction of CpG DNAs in C3H/HeJ mouse spleen cell cultures (an LPSlower-responsive strain). All three CpG DNAs tested in this assayinduced concentration-dependent cytokine secretion (FIGS. 20A and B).Since PO-CpG DNA 155 failed to induce cytokine secretion in BALB/c mousespleen cell cultures, it was not further tested in C3H/HeJ spleen cellcultures. Both PO-immunomers 156 and 157 induced higher IL-12 productionthan did PS-CpG DNA 4 (FIG. 20A). However, at concentrations up to 3μg/mL, neither induced IL-6 production. At the highest concentrationtested (10 μg/mL), both induced significantly less IL-6 than did PS-CpGDNA 4 (FIG. 20B). The ratio of IL-12 to IL-6 secreted is calculated todistinguish cytokine secretion profiles of PS and PO CpG DNAs (FIG. 20Ainset). In addition, the C3H/HeJ spleen cell culture results suggestthat the responses observed with CpG DNAs are not due to LPScontamination.

PS-CpG DNAs have been shown to induce potent antitumor activity in vivo.Since PO-CpG DNAs exhibited greater nuclease stability and inducedhigher levels of IL-12 and IFN-γ secretion in in vitro assays, we wereinterested to see if these desirable properties of PO-immunomers improvethe antitumor activity in vivo. We administered PO-immunomer 157subcutaneously at a dose of 0.5 mg/kg every other day to nude micebearing tumor xenografts of MCF-7 breast cancer cells that expresswild-type p53, or DU-145 prostate cancer cells that express mutated p53.PO-immunomer 157 gave 57% growth inhibition of MCF-7 tumors on day 15compared with the saline control (FIG. 21A). It also produced 52% growthinhibition of DU-145 tumors on day 34 (FIG. 21B). These antitumorstudies suggest that PO-immunomers of the proposed design exhibit potentantitumor activity in vivo.

Example 22 Short Immunomers

To test the effects of short immunomers on cytokine induction, thefollowing immunomers were used. These results show that immunomers asshort as 5 nucleotides per segment are effective in inducing cytokineproduction.

TABLE 22 Immunomer Structure and Immunostimulatory Activity in BABL/CMouse Spleen Cell Cultures Oligo Length/ IL-12(pg/mL) IL-6(pg/mL) OligoNo. Sequences and Modification (5′-3′) or Each Chain 10 μg/mL 10 μg/mL 4(SEQIDNO:2) 5′-CTATCTGACGTTCTCTGT-3′ 18mer 2731 4547  25(SEQIDNO:17)5′-CTATCTGTCGTTCTCTGT-3′ 18mer 795 789 158(SEQIDNO:13)

11mer 3490 5319 159(SEQIDNO:33)

11mer 3265 4625 160(N/A)

 6mer 2085 2961 161(N/A)

 6mer 3169 5194 162(N/A)

 6mer 1015 705 163(N/A)

 6mer 2623 3619 164(N/A)

 6mer 564 845 165(N/A)

 6mer 196 0 166(N/A)

 6mer 219 0 167(N/A)

 6mer 1441 5056 168(N/A)

 6mer 198 0 169(N/A)

 6mer 2410 4857 Normal phase represents a phosphorothioate linkage.

Equivalents

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. A method for generating an immune response in a vertebrate, themethod comprising administering to the vertebrate an immunomercomprising at least two oligonucleotides linked at their 3′ ends orinternucleotide linkages or a functionalized nucleobase or sugar to anon-nucleotidic linker, wherein at least one of the oligonucleotides isan immunostimulatory oligonucleotide having an accessible 5′ end andcomprising an immunostimulatory dinucleotide selected from the groupconsisting of C*pG, CpG* and C*pG*, wherein C is cytidine or2′-deoxycytidine, C* is 2′deoxythymidine, arabinocytidine,2′-deoxy-2′-substituted-arabinocytidine,2′-O-substituted-arabinocytidine, 2′-deoxy-5-hydroxycytidine,2′-deoxy-N4-alkyl-cytidine, 2′-deoxy-4-thiouridine or other non-naturalpyrimidine nucleoside, G is guanosine or 2′-deoxyguanosine, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′-substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, or other non-natural purinenucleoside, and p is an internucleoside linkage selected from the groupconsisting of phosphodiester, phosphorothioate, and phosphorodithioate.2. The method of claim 1 further comprising administering a vaccine. 3.The method of claim 2, wherein the immunomer or the vaccine, or both,are linked to an immunogenic protein.
 4. The method of claim 1 furthercomprising administering an adjuvant.
 5. The method according to claim1, further comprising administering an antigen.
 6. The method accordingto claim 1, wherein the non-nucleotidic linker is selected from thegroup consisting of a linker from about 2 angstroms to about 200angstroms in length, a metal, a soluble or insoluble biodegradablepolymer bead, an organic moiety having functional groups that permitattachment to the 3′-terminal nucleoside of the oligonucleotide, abiomolecule, a cyclic or acyclic small molecule, an aliphatic oraromatic hydrocarbon, either of which optionally can include, either inthe linear chain connecting the oligonucleotides or appended to it, oneor more functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthiourea; amino acids, carbohydrates, cyclodextrins, adamantane,cholesterol, haptens antibiotics, glycerol or a glycerol homolog of theformula HO—(CH2)_(o)—CH(OH)—(CH2)_(p)—OH, wherein o and p independentlyare integers from 1 to about 6, and a derivative of1,3-diamino-2-hydroxypropane.